Spindle pole body (SPB) duplication during the budding yeast cell cycle is a critical and essential event in assembly of the mitotic spindle that ultimately segregates the chromosomes to the progeny cells. In yeast, the SPB is the sole microtubule organizing center and is embedded in the nuclear envelope (NE) nucleating microtubules in the nucleoplasm and in the cytoplasm. Failure in SPB duplication or in SPB function leads to anueploidy and polyploidy similar to that observed in many cancer cells. This proposal addresses the function of two genes, NDC1 and MPS2, that are required during SPB duplication to insert the new SPB into the NE. We have found that these essential genes encode membrane proteins that localize to the SPB. In addition, Ndc1p is found in nuclear pore complexes (NPC) and Mps2p is found in the NE and the ER membranes. NPCs, like SPBs, are embedded in the NE.
The aims of this proposal are to first, explore the function of Ndc1p and Mps2p in SPB duplication by looking for genetic and physical interactions with other SPB components. We will also carry out genetic screens to look for additional genes that function in SPB duplication. Second, we will determine the topology of Ndc1p and Mps2p in the nuclear envelope. Furthermore, we will determine whether these proteins have access to the nucleoplasm and/or the cytoplasm as part of our efforts to determine how they function. Third, we will study the NPC-related function of Ndc1p. We believe that Ndc1p may be required for NPC assembly, like SPB assembly. We propose a variety of genetic, cytological and biochemical approaches to discover the role of Ndc1p at NPCs and to identify proteins with which it collaborates in this function. Finally, we have identified a novel gene, NOE1, that is required for the viability of ndc1-1 cells and whose product may be localized to the SPB, suggesting a possible role for Noe1p in SPB duplication. We will define the role of Noe1p with respect to Ndc1p, and identify genes that interact with NOE1. The proposed project is aimed at understanding the function of a set of unusual membrane proteins at the SPB and the NPC, and at identifying other proteins with which the interact at these organelles.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM059992-01A1
Application #
6128722
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Deatherage, James F
Project Start
2000-04-01
Project End
2004-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
1
Fiscal Year
2000
Total Cost
$216,936
Indirect Cost
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Boulder
State
CO
Country
United States
Zip Code
80309
Araki, Yasuhiro; Lau, Corine K; Maekawa, Hiromi et al. (2006) The Saccharomyces cerevisiae spindle pole body (SPB) component Nbp1p is required for SPB membrane insertion and interacts with the integral membrane proteins Ndc1p and Mps2p. Mol Biol Cell 17:1959-70
Lau, Corine K; Giddings Jr, Thomas H; Winey, Mark (2004) A novel allele of Saccharomyces cerevisiae NDC1 reveals a potential role for the spindle pole body component Ndc1p in nuclear pore assembly. Eukaryot Cell 3:447-58
Roberts, Paul; Moshitch-Moshkovitz, Sharon; Kvam, Erik et al. (2003) Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae. Mol Biol Cell 14:129-41
Giddings, T H (2003) Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples. J Microsc 212:53-61
McBratney, Susan; Winey, Mark (2002) Mutant membrane protein of the budding yeast spindle pole body is targeted to the endoplasmic reticulum degradation pathway. Genetics 162:567-78
O'Toole, Eileen T; Winey, Mark; McIntosh, J Richard et al. (2002) Electron tomography of yeast cells. Methods Enzymol 351:81-95
Giddings Jr, T H; O'Toole, E T; Morphew, M et al. (2001) Using rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis. Methods Cell Biol 67:27-42
Kerscher, O; Hieter, P; Winey, M et al. (2001) Novel role for a Saccharomyces cerevisiae nucleoporin, Nup170p, in chromosome segregation. Genetics 157:1543-53
Winey, M; O'Toole, E T (2001) The spindle cycle in budding yeast. Nat Cell Biol 3:E23-7
Gomez-Ospina, N; Morgan, G; Giddings Jr, T H et al. (2000) Yeast nuclear pore complex assembly defects determined by nuclear envelope reconstruction. J Struct Biol 132:5-Jan

Showing the most recent 10 out of 11 publications